Understanding the stability of DNA G-quadruplex units in long human telomeric strands

Abstract

Human telomeric DNA is composed of GGGTTA repeats. The presence of consecutive guanines makes the telomeric G-strand prone to fold into contiguous (or tandem) G-quadruplexes (G4s). The aim of this study was to provide a clarified picture of the stability of telomeric tandem G4 structures as a function of the number of G4 units and of boundary sequences, and an understanding of the diversity of their melting behaviors in terms of the single G4 units composing them. To this purpose we undertook an UV-spectroscopic investigation of the structure and stability of telomeric repeats potentially able to fold into up to four contiguous G4s, flanked or not by TTA sequences at their 5' and 3' extremities. We explain why the stability of (GGGTTA)4m-1GGG structures (m = 2, 3, 4 …) decreases with increasing the number m of G4 units, whereas the stability of TTA-(GGGTTA)4m-1GGG-TTA structures does not. Our results support that the inner G4 units have similar stabilities, whereas the stabilities of the terminal G4 units are modulated by their flanking nucleotides: in a TTA-(GGGTTA)4m-1GGG-TTA tandem context, the terminal G4 units are roughly as stable as the inner G4 units; while in a (GGGTTA)4m-1GGG tandem context, the G4 at the 5' extremity is more stable than the G4 at the 3' extremity, which in turn is more stable than an inner G4. Our study provides new information about the global and local stability of telomeric tandem G4 structures under near physiological conditions.

The binding efficiency of RPA to telomeric G-strands folded into contiguous G-quadruplexes is independent of the number of G4 units

“…S2) further suggest a poorly cooperative binding mode of hRPA to H51, H75 and H99, as previously observed with long nonstructured single-stranded oligonucleotides [30,32]. The higher hRPA binding efficiency ([hRPA] 50 around 5 nM) toward multimeric G4s compared to the single G4 ([hRPA] 50 around 20 nM) can be explained by the lower stability of these higher-order structures (temperature of thermal transition T t 50 C [26], Supplementary Information, Table S1) compared to the single G4 formed by H27 (T t 55 C [26], Supplementary Information, Table S1). Indeed, we previously reported an inverse relationship between the efficiency of binding of hRPA to a G4 sequence and the stability of the formed G4 [23].…”

“…In H69-Q1, the fluorophores are placed at both sides of the first G4 unit; in H69-Q2, they are placed at both sides of the second G4 unit; in H69-Q3, they are placed at both sides of the third G4 unit (Supplementary Information, Table S1). The doubly labeled G4 units of these three constructs have temperatures of half-denaturation in the range of 50e60 C, as previously assessed by melting experiments followed by fluorescence [26]. For all H69-Q1,2,3, fluorimetric titration with hRPA revealed an increase of FAM fluorescence and a decrease in TAMRA fluorescence upon hRPA addition, indicating that hRPA is able to open each of the three G4 units, independently of its position (Fig.…”

“…. We previously characterized the structures formed by these oligonucleotides: we showed that H51, H75 et H99 fold into two, three and four contiguous G4 units, respectively, which are non-interacting and have identical stabilities; while the single G4 formed by the shortest sequence H27 has a higher stability [26] (Supplementary Information, Table S1). We point out that the binding constant of hRPA to dT 50 , dT 60 and dT 70 oligonucleotides (longer than the size of the 30-nt RPA extended-binding mode and able in principle to bind more than one RPA, up to three RPA for dT 70 ) does not depend on their length [30].…”

“…The independence of hRPA binding efficiency of the number of contiguous G4 units may result from the poorly cooperative binding mode of hRPA. We asked if this feature is also due to the peculiar character of multimeric G4 structures formed by telomeric G-strands in potassium, where the contiguous G4 units are non-interacting and have similar stabilities, disregarding of their number [26]. To answer to this question, we studied the binding of hRPA to H21 ((G 3 T 2 A) 3 15 G 3 ) sequences in sodium, folding into one, two, three and four G4 units, respectively [34], but lacking the T 2 A tails at their extremities.…”

Structure, properties, and biological relevance of the DNA and RNA G-quadruplexes: Overview 50 years after their discovery

“…Furthermore, these multiquadruplex structures unfold at lower temperatures than individual G4s [193]. It was shown in 2015 why the stability of the multiquadruplex garland depends on the number of G quadruplex units [201]. The authors proved that the observed decrease in the stability of the structure formed by d((GGGT TA) 4m-1 GGG), where m = 2, 3, 4, with increase of the number of G4 units, was related to the fact that the G4 at the 5′ end of the molecule was more thermodynamically stable than the G4 at the 3′ end, whose stability in turn was higher than the stability of the inner quadruplex domains.…”